Ca2+-Camodulin dependent protein kinase II (CaMKII) is widely expressed in mammalian cells, with the d isoform predominantly expressed in cardiomyocytes. Our recent studies have shown that b1-adrenergic receptor stimulation induces cardiomyocyte apoptosis via PKA-independent CaMKII signaling. It is unknown, however, whether activation of CaMKII is sufficient to trigger cardiomyocyte apoptosis and if so, what is the underlying mechanism. To specifically manipulate CaMKII-dC activity, we overexpressed a constitutively active mutant of CaMKII-dC (CA-CaMKII-dC) using adenoviral gene transfer (m.o.i. 100) in cultured adult rat cardiomyocytes where CaMKII activity was increased by 2 fold after 24 h infection. Using Western blotting and confocal immunofluorescence imaging, we observed overexpressed CaMKII-dC in mitochondria as well as cytosol. In the infected cardiomyocytes, apoptosis occurred in a time-dependent manner. This was evidenced by CA-CaMKII induced increase in TUNEL positive staining cells and DNA fragmentation detected with DNA ladder. Furthermore, inhibition of CaMKII with KN-93 (1 mM) or a membrane permeable peptide inhibitor AIP (5 mM), or by adenoviral gene transfer of a dominant negative-CaMKII-dC (m.o.i. of 100) fully prevented CA-CaMKII-dC mediated apoptosis, indicating that enhanced CaMKII activation is sufficient to trigger cardiomyocyte apoptosis. More importantly, CaMKII-induced apoptosis was accompanied by increased cytochrosome C release from mitochondria in cardiomyocytes infected by Adv-CA-CaMKII-dC, suggesting that enhanced CaMKII signaling leads to the activation of mitochondrial apoptotic pathway. Thus, we conclude that sustained activation of CaMKII is sufficient to trigger cardiomyocyte apoptosis likely via a mitochondrial apoptotic pathway.